Summary – Project 4 Medulloblastoma encompasses four molecular subtypes that have different prognoses (WNT, Sonic Hedgehog [SHH], Group-3 and Group-4). Despite these differences, all children with medulloblastoma receive the same surgery, radiation, and chemotherapy. This treatment fails to cure most cases of Group-3 disease, and inflicts debilitating long-term side effects on children with WNT-medulloblastoma. Therefore, future efforts to cure all children with medulloblastoma must provide an understanding of disease biology that can guide the development of curative, relatively non-toxic, subtype-specific therapies. During the last funding cycle we focused on understanding WNT-medulloblastoma, complementing studies by our P01 colleagues of the other disease subtypes. We identified progenitor cells of the lower rhombic lip as the origin of WNT- medulloblastoma, and generated the first mouse model of this disease subtype. In addition, using whole genome sequencing (WGS), we identified over 40 novel mutations in medulloblastoma, including highly recurrent mutations in a new candidate oncogene of WNT and SHH-tumors, DDX3X. The proposed studies will build on these data, and through three new Specific Aims will address our central hypothesis: `Medulloblastoma subtypes are driven by distinct cell signals that can be targeted for therapeutic gain.' Through a comprehensive series of in vitro and in vivo phenotype and tumorigenesis assays, Aim 1 will determine the role of DDX3X in hindbrain development and medulloblastoma. Aim 2 will employ an innovative, multiplatform approach that integrates high-throughput drug screening, cell biology assays, and genomics to pinpoint key molecular therapeutic targets and matched inhibitors of WNT-medulloblastoma, including DDX3X. Aim 3 will test the subtype-specificity of potential new therapies identified in Aim 2 in the context of novel, combination, neurosurgical, irradiation, and chemotherapy trials in mice with WNT, SHH and Group-3 tumors. In this manner we will perform the most rigorous preclinical testing of new subtype-specific treatments of medulloblastoma to date, and thereby optimize the selection of combination treatments for translation to clinical trial.